Smart Microneedle Arrays Integrating Cell‐Free Therapy and Nanocatalysis to Treat Liver Fibrosis

Abstract Liver fibrosis is a chronic pathological condition lacking specific clinical treatments. Stem cells, with notable potential in regenerative medicine, offer promise in treating liver fibrosis. However, stem cell therapy is hindered by potential immunological rejection, carcinogenesis risk, efficacy variation, and high cost. Stem cell secretome‐based cell‐free therapy offers potential solutions to address these challenges, but it is limited by low delivery efficiency and rapid clearance. Herein, an innovative approach for in situ implantation of smart microneedle (MN) arrays enabling precisely controlled delivery of multiple therapeutic agents directly into fibrotic liver tissues is developed. By integrating cell‐free and platinum‐based nanocatalytic combination therapy, the MN arrays can deactivate hepatic stellate cells. Moreover, they promote excessive extracellular matrix degradation by more than 75%, approaching normal levels. Additionally, the smart MN arrays can provide hepatocyte protection while reducing inflammation levels by ≈70–90%. They can also exhibit remarkable capability in scavenging almost 100% of reactive oxygen species and alleviating hypoxia. Ultimately, this treatment strategy can effectively restrain fibrosis progression. The comprehensive in vitro and in vivo experiments, supplemented by proteome and transcriptome analyses, substantiate the effectiveness of the approach in treating liver fibrosis, holding immense promise for clinical applications.


Uptake of SecNPs by Cells
Stem cell secretome-encapsulated core-shell nanoparticles (SecNPs) were labeled with DiI by incorporating this dye into the oil phase during the preparation of SecNPs.Subsequently, the pre-cultured cells were co-incubated with DiI-labeled SecNPs for 0, 2, 4, or 6 h.After washing with PBS, the cell nuclei were stained with DAPI, followed by the fixation with 4% (w/v) paraformaldehyde (PFA, pH 7.4) at room temperature for 15 min.After another round of washing, the stained cells were observed and imaged with an inverted fluorescence microscope (Ti2-U, Nikon, Japan), and the DiI + fluorescence intensity was determined using a flow cytometer (CytoFLEX S, Beckman, USA).

Investigation of CAT-Like Activity of PtNZs
The catalase (CAT)-like activity of platinum-based nanozymes (PtNZs) was assessed through sequential H2O2 depletion and O2 generation assays, following the methods outlined in our previous study. [1]Amplex red assay was used to confirm the H2O2 consumption by PtNZs.
The reaction was continued for an additional 5 min after the addition and thorough mixing of Amplex red (25 μM) and HRP (7 U mL -1 ).Then, an ultraviolet-visible (UV-vis) spectral scanning from 450 nm to 650 nm was expeditiously conducted.
The monitoring of O2 generation resulting from the decomposition of H2O2 was further used to assess the CAT-like activity of PtNZs.Specifically, different concentrations of PtNZs (0, 2, 4, or 8 μg mL -1 ) were mixed with H2O2 (50 mM) in 100 mM PBS (15 mL, pH 7.4).The realtime measurement of O2 generation was conducted using a pen-type dissolved oxygen meter (P6345-01, I-Quip, China).

Investigation of SOD-Like Activity of PtNZs
The superoxide dismutase (SOD)-like activity of PtNZs was assessed using the NBT method, as described in our earlier study. [1]In detail, the riboflavin solution (20 μM), NBT solution (75 μM), methionine solution (13 mM), and PtNZ solution (0, 20, 40, 60, or 80 μg mL -1 ) were homogeneously mixed in Eppendorf (EP) tubes.PBS was supplemented to ensure equal total volume of solution in each EP tube.After exposure to UV light (254 nm) for 5 min, a rapid UV-Vis spectral scanning from 300 nm to 800 nm was performed, with peak absorption observed at 560 nm.The SOD-like activity of PtNZs is inversely proportional to the intensity of blue color of the reaction solution.

Intracellular Antioxidant Efficiency of PtNZs
The cell-permeable DCFH-DA was applied to indicate the ROS levels in hepatocytes.AML12 or LO2 cells were first cultured in a 24-well plate (2 × 10 4 cells well -1 ) for 24 h, followed by the co-incubation with PtNZs (0, 2, 4, or 8 μg mL -1 ) for 12 h.Subsequently, all cells were exposed to H2O2 (500 μM) for an additional 12 h.After washing with PBS, the treated AML12 or LO2 cells were stained with DCFH-DA at 37 °C for 30 min.Following an additional round of washing, the fluorescence images were captured, and the flow cytometry analysis was performed to quantify the results.

Intracellular O2 Generation of PtNZs
[Ru(dpp)3]Cl2, an O2-indicated fluorescence probe, was used to determine the O2 generation level in HSCs.LX2 or HSC-T6 cells were initially seeded in a 12-well plate (5 × 10 4 cells well - 1 ) and cultured for 12 h.Then, all cells were transferred into a hypoxic environment (1% O2) created in a small tris-gas incubator (Galaxy 48 R, Eppendorf, Germany), followed by another 12 h of culture.After that, the cells were co-incubated with PtNZs (0, 2, 4, or 8 μg mL -1 ) under hypoxic conditions for 12 h.The uninternalized PtNZs were removed by washing.Subsequently, the washed cells were concurrently co-incubarted with H2O2 (500 μM) and [Ru(dpp)3]Cl2 in hypoxic environment for 4 h.Lastly, the fluorescence images were captured, and the flow cytometry analysis was performed.

Extraction and Isolation of SPI
Soy protein isolate (SPI) was extracted and isolated according to the methods described in our previous report. [2]Briefly, the defatted soy meal underwent further grinding, defatting, and drying processes before being completely dispersed in ultrapure water.The initial pH of the dispersion was adjusted to 8.0 using a NaOH solution, followed by continuous agitation for 2 h, during which the pH was readjusted to 8.0 every 30 min using a 1 M NaOH solution.The pH of supernatant obtained by centrifugation at 8 000 g was adjusted to 4.5 by adding a HCl solution.Then, the precipitate was gathered via centrifugation at 5 000 g, followed by redispersion in ultrapure water.A clear crude protein solution was obtained after gradually adjusting the pH to 7.0, and subsequently transferred into dialysis tubes with a molecular weight cut-off range of 8-14 kDa.Following thorough dialysis in vigorously stirred ultrapure water, the resulting dialysate was collected and subjected to lyophilization to obtain SPI powder.

Effect of SPI on Hepatocytes and Injured Hepatocytes
LO2 cells were initially cultured in a 96-well plate (5 × 10 3 cells well -1 ) for 24 h.To simulate hepatocyte injury, the cells were treated with 500 μM H2O2 for 12 h.After washing with PBS, the normal or injured LO2 cells were co-incubated with 0.75% (w/v) SPI or NPr-hydrolyzed SPI for 24 h.Finally, the cell viability was assessed using a CCK-8 assay.

Responsive Degradation and Drug Release of MN Arrays
To evaluate the in vitro release property and predict the in vivo drug release behavior of microneedle (MN) arrays, Nile blue (NB) was encapsulated into MNs as a drug surrogate.The NB-loaded MNs were immersed into PBS (pH 7.4) at 37 °C under dark conditions.At predetermined time intervals, 100 μL of release medium (PBS) was collected, and an equal volume of fresh PBS was supplemented.After 1 h of release monitoring, a subset of the samples were subjected to heating at 45 °C for 5 min in a water bath to simulate the in vivo photothermal effect induced by NIR irradiation.The degradation of MN arrays and the release of drugs were continuously monitored during the experiment by quantifying the fluorescence intensity (excited at 630 nm) of the release medium using a fluorescence spectrophotometer (RF6000, Shimadzu, Japan).After obtaining a standard curve correlating the concentration of NB in PBS with fluorescence intensity, the calculation of cumulative release rate was performed.

Transcriptome Analysis of Liver Tissues
Transcriptome analysis was performed on liver tissues from the control and PSMN+NIR (treatment through PtNZ+SecNP-loaded MN patch implantation and NIR irradiation) groups.

Histologic Evaluation
The sampled liver tissues were fixed in 4% (w/v, pH 7.4) PFA solutions at room temperature for at least 1 day.Subsequently, the fixed samples were dehydrated using gradient ethanol solutions and finally embedded in paraffin.The embedded tissues were sectioned into slices with a constant thickness of 6 μm employing a paraffin microtome (RM 2235, Leica, Germany).
Subsequently, the liver tissue sections were thoroughly washed and co-incubated with goatderived anti-rabbit secondary antibodies conjugated with Alexa Fluor 488 (GB25303, Servicebio) or Cyanine 3 (GB21303, Servicebio) in the dark.Following another round of washing, the tissue sections were mounted using an antifade medium containing DAPI.Finally, bright-field and fluorescence images of stained tissue sections were visualized and captured utilizing an inverted fluorescence microscope (Ti2-U, Nikon) and analyzed quantitatively using Fiji software (version 1.53c, USA).

Biosafety Evaluation
To evaluate the safety of our implemented treatments, we utilized an automated biochemical analyzer (3100, Hitachi, Japan) to conduct serological testing on serum samples obtained by centrifuging blood at 4 °C for 20 min at a speed of 1 000 g. Additionally, histological analyses of harvested organs were performed using H&E staining and microscopic examination.

Figure S1 .
Figure S1.Fabrication of SecNPs.The conditioned medium of hUCMSCs, devoid of serum, was collected and subjected to centrifugation, filtration, and lyophilization to obtain the secretome powder.Subsequently, the secretome was redissolved and encapsulated within the PLGA core of SecNPs employing double emulsification and solvent evaporation techniques.

Figure S2 .
Figure S2.Heatmap illustrating all proteins and their intensities in hUCMSC secretome, analyzed using label-free quantitative methods.

Figure S3 .
Figure S3.Enrichment analysis of gene ontology (GO) terms associated with all proteins in hUCMSC secretome, providing insights into their biological processes, cellular components, and molecular functions.

Figure S4 .
Figure S4.Enrichment analysis performed on Kyoto encyclopedia of genes and genomes (KEGG) pathways of all proteins in hUCMSC secretome.

Figure S7 .
Figure S7.Uptake of SecNPs by hepatocytes.a-d) Internalization of DiI-labeled SecNPs by AML12 (a, b) and LO2 (c, d) cells illustrated by representative flow cytometry histograms (a, c) and representative fluorescence images with corresponding quantified 3D surface plots (b, d) over various co-incubation time periods.

Figure S8 .
Figure S8.Influence of pre-heating on the protective effect of SecNPs against the CCl4-induced hepatocyte damage in vitro.a, b) Cell viabilities of AML12 (a) and LO2 (b) cells, which were exposed to CCl4 and subsequently treated with SecNPs after being pre-heated at 45 °C for 30 min.Data are presented as mean ± SD (n = 3).Statistical significances were estimated employing one-way ANOVA followed by Tukey's multiple comparisons post hoc test.**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and n.s., not significant.

Figure S9 .
Figure S9.CAT-like activity of PtNZs evidenced by the of H2O2 consumption over various catalysis time periods.

Figure S12 .
Figure S12.In vitro protective effect of SPI in the thermal-responsive MNs.a) Cell viabilities of LO2 cells following the co-incubation with or without native SPI.b) Cell viabilities of LO2 cells sequentially undergoing the treatment with H2O2, and native SPI or NPr-hydrolyzed SPI.Data are shown as mean ± SD (n = 5).Statistical significances were evaluated by one-way ANOVA with Tukey's multiple comparisons post hoc test.*, P < 0.05; ****, P < 0.0001; and n.s., not significant.

Figure
Figure S13.a) Schematic illustration of fabricating MN arrays.b) Representative visual appearance of a microneedle patch undergoing the mechanical compression test.

Figure S14 .
Figure S14.Photothermal effect of PMNs.a) Photothermal temperature curves of PMN arrays under NIR irradiation at varying power levels.b) Representative infrared thermal images of PMN arrays containing different concentrations of PtNZs.

Figure S15 .
Figure S15.Surgical implantation and treatment with MN arrays.a) Representative digital image capturing the process of laparotomy and implantation of an MN patch in a mouse.b) Representative infrared thermal image of the mouse post-implantation of the MN arrays containing PtNZs during exposure to NIR irradiation, indicating the in situ photothermal effects.

Figure S16 .
Figure S16.In vivo analysis of proliferation and apoptosis in liver tissues.Representative Ki67 + (indicating cellular proliferation) immunofluorescence and TNUEL + (reflecting apoptosis) fluorescence images of liver tissues from the normal, liver fibrosis (LF), and PSMN+NIR groups.

Figure S18 .
Figure S18.Biosafety determined through the histological evaluation.Representative H&Estained images of hearts from mice in the indicated groups.

Figure S19 .
Figure S19.Biosafety determined through the histological evaluation.Representative H&Estained images of spleens from mice in the indicated groups.

Figure S20 .
Figure S20.Biosafety determined through the histological evaluation.Representative H&Estained images of lungs from mice undergoing the designated treatments.

Figure S21 .
Figure S21.Biosafety determined through the histological evaluation.Representative H&Estained images of kidneys from mice in the designated groups.

Figure S23 .
Figure S23.Enrichment analysis performed on GO terms of DEGs in the liver tissues from LF group and normal group.a) GO terms associated with biological process.b) GO terms implicated in cellular component and molecular function.

Figure S24 .
Figure S24.Enrichment analysis performed on KEGG main classes and pathways of DEGs in the liver tissues from LF group and normal group.a) Percent of DEGs enriched in various pathways assigned to different KEGG main classes.b) Rich factor of DEGs in different KEGG pathways.

Figure S25 .
Figure S25.Enrichment analysis conducted on GO terms of DEGs in the liver tissues from PSMN+NIR group and LF group.

Figure S26 .
Figure S26.Enrichment analysis performed on KEGG main classes and pathways of DEGs in the liver tissues from PSMN+NIR group and LF group.a) Percent of DEGs enriched in various pathways assigned to different KEGG main classes.b) Rich factor of DEGs in different KEGG pathways.

Figure S27 .
Figure S27.GSEA based on Reactome enrichments of DEGs in liver samples from LF group and normal group, including upregulated (P < 0.05 and NES > 1) and downregulated (P < 0.05 and NES < -1) pathways involved in ECM deposition and degradation, and HSC activation and quiescence.

Table S1
Primer sequences used in RT-qPCR assays.